Influence of senescence and of carbohydrate levels on the pattern of leaf proteases in purple nutsedge (Cyperus rotundus)

Cyperus rotundus L. is a monocotyledonous perennial weed, which forms large numbers of tubers during its vegetative growth. Since these tubers represent major sinks, source/sink interactions are more complex, and leaf senescence and proteolytic processes in this species may be different from the situation in the well‐investigated annual crop plants characterized by monocarpic senescence. Judged by native PAGE and by inhibitor studies, three different aminopeptidases, one iminopeptidase, two or more carboxypeptidases and two or more different endopeptidases were present in mature green leaves. Exo‐ and endoproteolytic activities increased during the senescence of excised leaf segments. A marked change was observed in the endopeptidase pattern, since a cysteine proteinase activity was strongly induced during senescence of the segments. This endopeptidase was also found in naturally senescing leaves and may, therefore, participate in nitrogen salvage from these organs. An increase of different protease activities was demonstrated in leaf segments of C. rotundus in the presence of high carbohydrate levels. The mechanisms involved, and the importance of this phenomenon for the interaction between source/sink relations and senescence, remain to be demonstrated.     

Degradation of tobacco pathogenesis-related proteins : evidence for conserved mechanisms of degradation of pathogenesis-related proteins in plants

Tobacco (Nicotiana tabacum L.) leaves were found to contain an extracellular proteinase that endoproteolytically cleaves tobacco pathogenesis-related (PR) proteins. This proteinase was partially purified from tobacco leaves and characterized as an aspartyl proteinase with a pH optimum around pH 3 and a molecular mass of 36,000 to 40,000 daltons. In vitro, the enzyme cleaved purified tobacco and tomato PR proteins into discrete fragments. The characteristics of this proteinase were similar to pepsin and identical to those displayed by a previously described tomato 37-kilodalton aspartyl proteinase active against tomato PR proteins (I Rodrigo, P Vera, V Conejero [1989] Eur J Biochem 184: 663-669), suggesting that these extracellular proteases could play a role in a conserved mechanism for PR protein turnover in plants.     

Immunological Identification of Proteinase Inhibitors I and II in Isolated Tomato Leaf Vacuoles

Proteinase inhibitor I has been identified and quantified in isolated vacuoles from tomato (Lycopersicon esculentum) leaves induced to accumulate inhibitors either by wounding or by supplying excised leaves with the wound hormone, proteinase inhibitor-inducing factor. Proteinase inhibitor II was also identified in the vacuoles but not quantified. Control vacuoles were prepared from unwounded plants that did not contain inhibitors. Vacuole to leaf cell ratios of inhibitors, chlorophyll, and several vacuolar and cytoplasmic enzymes were determined. The inhibitors were found almost entirely in the vacuoles. Acid phosphatase was located in control leaf vacuoles, but was found in both vacuoles and cytoplasm in induced leaves. Carboxypeptidase, induced by wounding, was found distributed between the vacuoles and cytoplasm of induced leaves. Low vacuole to leaf cell ratios of three cytoplasmic markers, triosephosphate isomerase, catalase, and chlorophyll, indicated that the isolated vacuoles were relatively free of intact protoplasts and cell debris.     

Proteinase Inhibitor-inducing Factor in Plant Leaves: A Phylogenetic Survey

Thirty-nine plant species representing 20 families from the four major divisions of plants were surveyed for the presence of proteinase inhibitor-inducing factor activity in leaves or other tissues. Tissue juices were assayed for their capacity to induce accumulation of proteinase inhibitor I in excised tomato (Lycopersico esculentum) leaves. In tissues of only 2 of the 39 species was proteinase inhibitor-inducing factor-like activity not found. The activity was absent in cabbage leaves and celery stalks. Fruiting bodies from one of three fungi genera assayed contained exceptionally large quantities of proteinase inhibitor-inducing factor-like activity. Extracts from Agraricus campestris fruiting bodies contained over 20 times more activity than tomato leaf juice. The survey confirms that substances with proteinase inhibitor-inducing factor-like activity are widespread in the plant kingdom.     

Detection and evaluation of serine proteinase by affinity chromatography on immobilized-aprotinin inRicinus communia

Neutral proteinase was found in the leaves ofRicinus communie as assayed with α-casein and H-D-Val-Leu-Lys-pNA as substrates. The enzyme is maximally active at pH around 7.4. A selective adsorbent for serine proteinase was prepared by attaching aprotinin to aminoalkyl-porous glass. When partially purified leaf proteinase was passed through a column containing this adsorbent, the proteinase activity present was bound to the porous glass. The proteinase eluted at IM NaCl was inhibited by aprotinin, leupeptin, DFP, phenylmethylsulfonyl fluoride (PMSF) and serine proteinase inhibitor fromR, communis leaves, whereas pepstatin, EDTA, EGTA, and DTT had no effect on the enzyme. This inhibition profile suggests the leaf proteinase is a neutral proteinase, such as a serine proteinase.     

Radial diffusion as a sensitive method for screening endopeptidase activity in plant extracts

The radial diffusion assay was evaluated for its usefulness as a simple, inexpensive assay for screening endopeptidase activity in vegetative plant tissues. Various substrates embedded in agar were tested with trypsin and α-chymotrypsin to select those with the greatest sensitivity for detecting proteolysis. Gelatin was the most sensitive substrate for assays and could be utilized to detect less than 1 ng of α-chymotrypsin and trypsin per 25 μl of solution. Several exopeptidases could not be detected by this method at 1000-fold higher concentrations of enzymes. Extracts of leaves of 17 genera representing 12 families were surveyed semiquantitatively for proteolytic activity. The leaves of genera of the Solanaceae, Leguminosae, Gramineae, and Compositae exhibited the highest levels of activity. Only leaves from Syringa vulgaris (Oleaceae) did not exhibit proteolytic activity with this assay. The method of radial diffusion was successfully employed for the initial concentration and purification of a proteinase from potato leaves.     

Proteinase activity in potato plants

Several vegetative tissues of potato plants were screened for proteinase activity. Both endopeptidase and exopeptidase activities were investigated using gelatin and L-amino acid-4-nitroanilides (benzoyl-L-arginine-4-nitroanilide/BAPA, glutaryl-L-phenyl-alanine-4-nitroanilide/GLUPHEPA, alanine-4-nitro-anilide/APA, leucine-4-nitroanilide/LPA, and benzoyl-L-tyrosine-4-nitroanilide/BTPA) as substrates. Leaves and rootes were found to contain the highest levels of endopeptidase activity; lesser activities were detected in flower petals, sprouts, and tubers. Three different types of proteinases, L-BAPAase (serine proteinase), APAase (thiol proteinase), and BTPAase (sensitive to reducing agents), were characterized in various physical and chemical properties. Their temperature optima were determined to be 25° (L-BAPAase) and 40° (BTPAase, APAase) respectively; their pH optimum was between 8.6 and 9.0, their isoelectric points were between pH 4.25 and 6.0, and their molecular weight was estimated 70,000 (L-BAPAase, APAase) and between 150,000–250,000 (BTPAase). The trypsin-like activity against L-BAPA was inhibited by diisopropylfluorophosphate and by tosyllysine-chloromethyl ketone, but not by trypsin inhibitors from potato and legume.Abbreviations APA alanine-4-nitroanilide - BAPA benzoyl-L-arginine-4-nitroanilide - BTPA benzoyl-L-tyrosine-4-nitroanilide - DFP diisopropylfluorophosphate - DMF dimethyl formamide - EDTA ethylenedinitrilotetraacetic acid - GLUPHEPA glutaryl-L-phenylalanine-4-nitroanilide - LPA leucine-4-nitroanilide - PHMB p-hydroxy-mercuribenzoate - PI-I potato chymotrypsin inhibitor I - PPI potato proteinase leaf - PPr potato proteinase root - PPt potato proteinase tuber - PVP polyvinylpyrrolidone - TLCK tosyl-L-lysinechloromethyl ketone - TPCK tosyl-L-phenylalanyl chloromethane     

Effect of changed source/sink relations on proteolytic activities and on nitrogen mobilization in field-grown wheat (Triticum aestivum L.)

Nitrogen mobilization and the pattern of proteolytic enzymeswere investigated in leaves and glumes of field-grown winterwheat (Triticum aestivum L.) during maturation. Source/sinkrelations were changed by removal of the ear, the flag leafor the lower leaves shortly after anthesis. Removal of the earwas most effective, resulting in delayed senescence of the flagleaf with the chlorophyll, aminopeptidase and carboxypeptidaseactivities remaining high in contrast to the control, whereasneutral endopeptidase activity increased more slowly. No majorchanges were observed in the second leaf from the top in plantswith either ears or flag leaves removed. Nitrogen mobilizationand proteolytic activities in glumes and the remaining leaveswere influenced only slightly by leaf removal. In earless plants,nitrogen was transported from the second leaf into the leafsheath and stem, but in the flag leaf the total reduced nitrogenremained high and free amino groups increased. The increase in endopeptidase activity was influenced by thesource/sink relations. However, the accumulation of amino groupsand the increasing endopeptidase activity in the flag leaf ofearless plants suggest that the nitrogen sink capacity did notgreatly control protein degradation; it remains to be seen whetherphytohormones, accumulated amino acids or other factors delayedthe increase in endopeptidase activity. (Received September 3, 1979; )     

The ADAXIALIZED LEAF1 gene functions in leaf and embryonic pattern formation in rice

The adaxial-abaxial axis in leaf primordia is thought to be established first and is necessary for the expansion of the leaf lamina along the mediolateral axis. To understand axis information in leaf development, we isolated the adaxialized leaf1 (adl1) mutant in rice, which forms abaxially rolled leaves. adl1 leaves are covered with bulliform-like cells, which are normally distributed only on the adaxial surface. An adl1 double mutant with the adaxially snowy leaf mutant, which has albino cells that specifically appear in the abaxial mesophyll tissue, indicated that adl1 leaves show adaxialization in both epidermal and mesophyll tissues. The expression of HD-ZIPIII genes in adl1 mutant increased in mature leaves, but not in the young primordia or the SAM. This indicated that ADL1 may not be directly involved in determining initial leaf polarity, but rather is associated with the maintenance of axis information. ADL1 encodes a plant-specific calpain-like cysteine proteinase orthologous to maize DEFECTIVE KERNEL1. Furthermore, we identified intermediate and strong alleles of the adl1 mutant that generate shootless embryos and globular-arrested embryos with aleurone layer loss, respectively. We propose that ADL1 plays an important role in pattern formation of the leaf and embryo by promoting proper epidermal development.     

Preparation of an exopeptidase-enriched fraction from the hepatopancreas of decapods

A laboratory-scale process suitable for the large-scale recovery of exopeptidases from the hepatopancreas tissue of decapod viscera is described. A method for distinguishing the relative exopeptidase and endopeptidase activities of digestive proteases was developed and used to monitor the enrichment of exopeptidases in an endo- and exopeptidase mixture. Simple extracts of the digestive tissues from decapods were a rich source of proteolytic activity; however, they contain a relatively high ratio of endopeptidase to exopeptidase activity. Based on the finding that most of the endopeptidase activity of extracts was inhibited by serine proteinase inhibitors, the relative exopeptidase activity of crayfish and langostilla crab digestive proteases was enriched by Agarose-SBTI affinity chromatography.     

Partial characterization of a protease inhibitor which inhibits the major endopeptidase present in the cotyledons of mung beans

Germination of mung beans (Phaseolus aureus, Roxb.) is accompanied by an increase in the activity of the endopeptidase involved in storage protein metabolism. Enzyme activity in the cotyledons increases 25-fold during the first 5 days of germination. The cotyledons also contain inhibitory activity against the endopeptidase, and this inhibitory activity declines during germination, suggesting that inhibitors may play a role in regulating the activity of the endopeptidase.     

丝氨酸内肽酶在黄瓜叶片衰老中的作用

采用丝氨酸内肽酶抑制剂和植物生长调节剂处理离体黄瓜叶片,研究了黄瓜叶片暗诱导衰老过程中丝氨酸内肽酶的作用。结果表明,6-BA50μmol／L与丝氨酸内肽酶抑制剂AEBSF能抑制叶片内肽酶活性的升高,延缓蛋白质降解,而ABA50μmol/L则促进了内肽酶活性的升高：其作用效果与AEBSF相反。活性电泳结果显示,黄瓜叶片中检测到6条内肽酶同工酶,其中4条（CEP2、3、4、6）为丝氨酸类型内肽酶,而ABA使丝氨酸内肽酶CEP2、3、4、6的活性明显增强,提示了丝氨酸类型内肽酶在黄瓜叶片衰老过程中具有重要作用。     

Condensed tannins of cotton leaves

Some primitive races of cotton (Gossypium hirsutum) are almost immune to spider mites. These strains contain condensed tannins of about 20% of dry wt. The tannins accumulate in leaves and their concentration increases in successive leaves until about the 10th true leaf. The upper leaves maintain this quantity until early fall. The condensed tannins give a moderate astringency to the leaf, and are mixed polymers hydrolysing in acidic digests to 1 part cyanidin and 4 parts delphinidin.     

Chloramphenicol Acetyl Transferase (CAT) Protein Is Expressed in Transgenic Tobacco in Field Tests following Attack by Insects

The expression of chloramphenical acetyl transferase (CAT) protein driven by the wound-inducible promoter from the proteinase inhibitor II K (pin2) gene was examined in whole tobacco (Nicotiana tabacum L.) plants under field conditions. Mechanical wounding of the field-grown leaves caused an accumulation of CAT protein in these leaves which begins several hours after wounding and continues to accumulate for about 36 hours. When sections of leaves were assayed for accumulation of CAT protein following wounding, the CAT protein was found to accumulate in the apical portions of the leaves. When endogenous insects attacked the leaves of transgenic plants grown in the field, the plants responded by inducing CAT protein. The mesophyll cells of the leaf were the site of expression of the CAT protein rather than the mid-vein or major veins within the leaf blade, indicating that the wound-inducible pin2 promoter specifically directs the synthesis of novel genes in tissues preferentially consumed by larval insects.     

五种黄精属植物的蛋白水解酶谱研究

用蛋白水解酶复性电泳方法 (G- PAGE)分析了 5种黄精属植物根状茎和叶的蛋白水解酶的种类和活性。结果表明 :(1)它们的根状茎均含有 85k D和 55k D的蛋白水解酶 ;叶均含有 82 k D的蛋白水解酶 ;(2 )根状茎和叶的蛋白水解酶种类和活性有很大差异 ,叶的蛋白水解酶活性为根状茎的 10倍 ,它们的活性均受 p H影响 ,其最适 p H为 7;(3)每种植物都含有自己特有蛋白水解酶 ;(4 )蛋白水解酶在植物鉴定中有参考价值     

In Vitro Stability and Inactivation of Peptide Hydrolases Extracted from Phaseolus vulgaris L.

Endopeptidase activity against azocasein had a higher temperatureoptimum (50°C) in leaf extracts than in cotyledon extracts(37°C). The temperature optima for aminopeptidase (46°C)and for carboxypeptidase (53°C) were similar in leaf andcotyledon extracts. The endopeptidase activity showed an excellentstability in crude extracts from leaves even at 37°C, whilethe endopeptidase in cotyledon extracts was less stable. Carboxypeptidasewas very stable in both leaf and cotyledon extracts. Aminopeptidasewas the least stable of the enzymes investigated and its inactivationrate depended on the source of the extract. A moderate stabilitywas observed in extracts of leaves or of ungerminated seeds,but this enzyme was rapidly inactivated in cotyledon extractsat pH 5.4. At pH 7.5 aminopeptidase remained active longer thanat pH 5.4. From experiments with mixed extracts it could beconcluded that in cotyledons an aminopeptidase inactivatingfactor was formed during germination. This factor was heat sensitive,excluded by Sephadex G-25, precipitated by 75% ammonium sulfateand inhibited by tosyl-L-lysine chloromethyl ketone. These datasuggest that the factor is a protein and considering the similarproperties it appears possible that it is the endopeptidaseformed during germination. (Received May 15, 1981; Accepted July 18, 1981)     

Oil body-mediated defense against fungi: From tissues to ecology

Oil bodies are localized in the seed cells and leaf cells of many land plants. They have a passive function as storage organelles for lipids. We recently reported that the leaf oil body has an active function as a subcellular factory that produces an antifungal oxylipin during fungal infection in Arabidopsis thaliana. Here, we propose a model for oil body-mediated plant defense. Remarkably, senescent leaves develop oil bodies and accumulate α-dioxygenase1 (α-DOX1) and caleosin3 (CLO3) on the oil-body membrane, which catalyze the conversion of α-linolenic acid to the phytoalexin 2-hydroxy-octadecatrienoic acid (2-HOT). The model proposes that senescent leaves actively produce antifungal oxylipins and phytoalexins, and abscised leaves contain a mixture of antifungal compounds. In natural settings, the abscised leaves with antifungal compounds accumulate in leaf litter and function to protect healthy tissues and young plants from fungal infection. Plants might have evolved this ecological function for dead leaves.     

Purification and characterization of a cysteine endopeptidase in cotyledons of germinated mung bean seeds

A cysteine endopeptidase (EC 3.4.22.-) present in cotyledons of mung bean (Vigna radiata) seedlings was purified to homogeneity, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This proteinase has an apparent molecular mass of 33 kilodaltons as estimated by SDS-PAGE and belongs to the class of cysteine proteinases as judged by the effects of various proteinase inhibitors on the activity of the enzyme. When proangiotensin is used as a substrate, the enzyme preferentially hydrolyzes the peptide bonds formed by the amino group of Leu or lle in this oligopeptide chain; for the enzyme to cleave those bonds, peptide sequences consisting of at least three amino acid residues on the amino side of Leu or lle must be present. The proteinase readily digests globulin present in mung bean cotyledons to smaller polypeptides.     

Hemoglobin-digesting Acid Proteinases in Soybean Leaves: CHARACTERISTICS AND CHANGES DURING LEAF MATURATION AND SENESCENCE

Three proteinases which digest hemoglobin rapidly at acid pH (3.5 to 4.5) were identified in crude extracts of soybean (Merr.) leaves and separated by chromatography on DEAE-cellulose. All three enzymes were endopeptidases as judged by the ratio of α-amino-nitrogen plus peptide nitrogen over α-amino-nitrogen in the trichloroacetic acid-soluble portion of hemoglobin digests. Proteinase I did not bind to diethylaminoethyl cellulose and was not inhibited by any of the proteinase inhibitors tested. Proteinase II was partially inhibited by phenylmethylsulfonyl fluoride, N-ethylmaleimide, and p-chloromercuribenzoate. The inhibition by phenyl-methylsulfonyl fluoride can probably be accounted for by the presence of contaminating carboxypeptidase. Proteinase III was the most anionic of the three and required the presence of sulfhydryl reagents to prevent the irreversible loss of activity. All the proteinase preparations digested soy-bean ribulose bisphosphate carboxylase as shown by the disappearance of the large subunit of that protein, when partially digested preparations were subjected to electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. These experiments confirmed that the three proteinases were endopeptidases. All three proteinases were present throughout leaf development; proteinase I predominated in expanding leaves, whereas proteinase III became the predominant enzyme as the leaves matured. Senescence (yellowing) was associated with a decline in the activities of all three proteinases.     

Barley proteinase inhibitors: A possible role in grasshopper control?

Young growing barley seedlings contain trypsin and chymotrypsin proteinase inhibitors in their leaf juices. The amount of chymotrypsin inhibitor varies greatly while trypsin inhibitor content is nearly the same in all varieties tested. Compana (CI5438) barley has much more total inhibitor than does Trebi (CI 936), Titan (CI 7055), Horn (CI 926), Hiproly (CI 3947) and Hiproly Normal (CI 4362). The distribution of inhibitors in different barley varieties correlates with the severity of grasshopper damage observed by other workers. Barley leaves could not be induced to accumulate proteinase inhibitors after excision and incubation, wounding, or absorption of crude “proteinase inhibitor inducing factor” preparations. Grasshopper damage experiments as related to proteinase inhibitor should be done in the field using yield as the correlative factor.